Steven J. Padula

The nuclear receptor PPAR gamma and immunoregulation: PPAR gamma mediates inhibition of helper T cell responses.

The peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors belonging to the nuclear receptor superfamily. Until recently, the genes regulated by PPARs were those believed to be predominantly associated with lipid metabolism. Recently, an immunomodulatory role for PPARγ has been described in cells critical to the innate immune system, the monocyte/macrophage. In addition, evidence for an antiinflammatory role of the PPARγ ligand, 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) has been found. In the present studies, we demonstrate, for the first time, that murine helper T cell clones and freshly isolated splenocytes express PPARγ 1. The PPARγ expressed is of functional significance in that two ligands for PPARγ, 15d-PGJ2 and a thiazolidinedione, ciglitazone, mediate significant inhibition of proliferative responses of both the T cell clones and the freshly isolated splenocytes. This inhibition is mediated directly at the level of the T cell and not at the level of the macrophage/APC. Finally, we demonstrate that the two ligands for PPARγ mediate inhibition of IL-2 secretion by the T cell clones while not inhibiting IL-2-induced proliferation of such clones. The demonstration of the expression and function of PPARγ in T cells reveals a new level of immunoregulatory control for PPARs and significantly increases the role and importance of PPARγ in immunoregulation.

Reactivity of dog sera to whole-cell or recombinant antigens of Borrelia burgdorferi by ELISA and immunoblot analysis.

Enzyme-linked immunosorbent assays (ELISAs) with separate preparations of 10 purified recombinant antigens of Borrelia burgdorferi sensu stricto were used to test sera from 36 dogs not vaccinated with whole cells of this agent and from five dogs vaccinated with whole-cell B. burgdorferi bacteria. All dogs lived in tick-infested areas of Connecticut and south-eastern New York state, USA. The non-vaccinated dogs had limb or joint disorder, lameness and fever during the period 1984-1991 and had antibodies to B. burgdorferi, as determined by a polyvalent ELISA with whole-cell antigen. In re-analyses of sera for total immunoglobulins in ELISAs with recombinant antigens, reactions were most frequently recorded when outer-surface protein (Osp) F, protein (p)35, p37, p39 and p-41G (a flagellin component) were tested separately. Western immunoblots of a subset of 16 sera, positive by ELISA with whole-cell antigen and representing a range of antibody titres (640-40960), verified immune responses to these or other lysed whole-cell antigens. Sera from vaccinated dogs contained antibodies to OspA, OspB, p22, p37 and p41-G. Therefore, serological reactions to OspF, p35 and p39 were the most important indicators of natural exposure to B. burgdorferi. Serum reactivities to these recombinant antigens in ELISAs can be used to help identify possible natural infections of canine borreliosis in dogs not vaccinated with whole-cell B. burgdorferi and to provide information on the geographic distribution of this bacterium.

Maintenance of antigen specificity by human interleukin-2-dependent T cell lines. Use of antigen-presenting cells and OKT3 antibody in the absence of antigen.

The in vitro growth of T cells obtained from localized anatomic sites of pathology may offer a new approach to the investigation of certain human autoimmune diseases. However, if interleukin-2-dependent T cell cloning is to be useful in helping to elucidate putative pathogenetic antigens in these diseases, the expansion of the small number of T cells obtainable from localized anatomic sites of pathology will often have to be accomplished in the absence of these, as yet undetermined, antigens. At present, it is a generally held belief that antigen-responsive, interleukin-2-dependent T cell lines and clones will lose antigen responsiveness if propagated in the absence of specific antigen. Thus, the use of T cell cloning might be viewed as being of limited usefulness in the investigation of certain human autoimmune diseases. In this report we demonstrate that, when propagated in the absence of antigen, human tetanus toxoid-specific, interleukin-2-dependent T cell lines will indeed lose antigen reactivity. However, if propagated in the absence of antigen but in the presence of antigen-presenting cells, the tetanus toxoid reactivity of a subset of such lines can be maintained. Moreover, the propagation with OKT3 antibody, in addition to antigen-presenting cells, may be even more effective in maintaining antigen reactivity. These results may suggest a new approach to the use of T cell cloning technology in the investigation of certain autoimmune diseases.

Cell-mediated immunity in rheumatic disease

Rheumatoid arthritis, systemic lupus erythematosus, and systemic sclerosis are the three most common systemic rheumatic diseases in which disordered immune function is thought to play a pathogenetic role. Each disease has different and characteristic abnormalities of the cellular immune system. In rheumatoid arthritis the identified abnormalities of immunoregulation are largely limited to specific antigens: Epstein-Barr virus and collagen. Systemic lupus erythematosus is characterized by exuberant B-cell activity with exaggerated humoral response, a diversity of autoantibodies, non-antigen-specific loss of suppressor cell function, and general suppression of cell-mediated immunity. In systemic sclerosis systemic defects of cellular and humoral immune function are mild, but the release of lymphokines and monokines at sites of inflammatory lesions is thought to be important in the pathogenesis of the disease. Similar immune cell-connetive tissue cell interactions are probably important in the propagation of rheumatoid synovitis. Thus, despite the many shared clinical and serologic features of these diseases as well as the presence of many patients who have clinically overlapping features of more than one of these entities, the immune defects and the immunopathogenesis of these disorders appear to be distinct.

Presentation of synthetic peptide antigen encoded by the MAGE-1 gene by granulocyte/macrophage-colony-stimulating-factor-cultured macrophages from HLA-A1 melanoma patients

The recent identification of the sequences of the peptides derived from a number of human melanomaassociated antigens has presented opportunities for developing a specific-peptide-based vaccine in this form of cancer. Since antigen-presenting cells (APC) play a crucial role in the induction of the T-cell-mediated immune response, we examined whether or not ex vivo cultured APC, bearing the appropriate MHC restricting elements, when pulsed with a relevant melanoma-specific cytotoxic-T-lymphocyte(CTL)-determined peptide, can present the peptide to the CTL. Here we show that a population of cells, derived from the monocyte/macrophage lineage from peripheral blood and grown in granulocyte/macrophage-colony-stimulating factor, exhibit many essential characteristics of “professional” APC (dendritic-type morphology with a proportion of the population, the B7 molecule, and high levels of MHC class I and class II molecules, CD11b and CD54 molecules) and are capable of efficiently presenting the nonapeptide, EADPTGHSY, encoded by the melanoma antigen MAGE-1 gene, to the MAGE-1-specific CTL clone, 82/30. These results suggest that this type of autologous ex vivo cultured population of professional APC, when pulsed with the relevant-CTL-determined peptide, can serve as a novel type of candidate vaccine for active specific immunization against HLAA1-positive patients with melanoma expressing the MAGE-1 antigen.

Effects of H-7 are not exclusively mediated through protein kinase C or the cyclic nucleotide-dependent kinases.

Culturing murine T cell tumor lines in the presence of the protein kinase inhibitor H-7 for 4 days led to their dependence on H-7 for maximal constitutive proliferation. Withdrawal of H-7 from H-7-conditioned cells led to inhibition of proliferation and cell death. The mechanism underlying this H-7 dependence does not appear to be related to clonal selection or to effects on protein kinase C or the cyclic nucleotide-dependent kinases. This suggests that all the effects of the widely used H-7 may not be completely understood, and that H-7 may be useful in the dissection of the complex patterns of growth regulation in T cell malignancies.

Analysis of cytolytic effector cell response in vitro against autologous human tumor cells genetically altered to synthesize interleukin-2

Recently, there has been a surge of interest in gene therapy in cancer particularly with cytokine transduced tumor cells as a novel form of tumor vaccine. In two autologous human tumor systems, using the tumor cells engineered to produce interleukin-2 by gene transduction techniques, we have examined whether or not such genetically altered cells are capable of inducing a tumor specific cytolytic T cell (CTL) response, in vitro, in co-culture with the respective autologous peripheral blood lymphocytes (PBL). We found that in neither system did co-cultures of the IL-2 producing tumor cells and the autologous PBL generate much cytolytic effector cell activity directed against the respective tumor cells, although these co-cultures did lead to the generation of substantial levels of natural killer (NK) cell activity when measured against the prototype NK sensitive target K562 line. More surprisingly, the levels of lymphokine activated killer cell responses against the respective autologous targets that could be generated in the PBL with exogenous IL-2 alone were compromised by the presence of the autologous tumor cells in the co-culture.

T cell receptor beta chain gene rearrangement shared by murine T cell lines derived from a site of autoimmune inflammation.

Advances in our understanding of the structure and molecular biology of the T lymphocyte antigen-receptor have now made it feasible to study human autoimmune diseases using new approaches. One such approach involves cloning of T cells from sites of autoimmune pathology followed by identification of putative disease-related T cell oligoclonality at the level of the T cell receptor gene rearrangements. We have now tested the feasibility of this approach in an animal model of autoimmunity, murine experimental allergic encephalomyelitis (EAE). Spinal cord-derived, self (murine) myelin basic protein (MBP)-reactive T cell lines and sublines were analyzed at the level of their receptor beta chain rearrangements using Southern blots. We now report that the MBP-reactive T cell lines and sublines derived from the spinal cords of four of five SJL/J mice with EAE share a 14.5-kb rearranged T cell receptor beta 1 band on Southern blots. A spinal cord-derived T cell line that was reactive to purified protein derivative of tuberculin (PPD), several lymph node-derived ovalbumin- and PPD-reactive T cell lines, as well as one MBP-reactive spinal cord-derived T cell line did not share this 14.5-kb rearranged beta 1 band. These results suggest that analysis of the antigen receptors used by T cells cloned from sites of inflammation may be a useful initial approach for identifying pathogenetically relevant T cells in the study of certain human autoimmune diseases.